Absence of NR2E1 mutations in patients with aniridia.

Purpose Nuclear receptor 2E1 (NR2E1) is a transcription factor with many roles during eye development and thus may be responsible for the occurrence of certain congenital eye disorders in humans. To test this hypothesis, we screened NR2E1 for candidate mutations in patients with aniridia and other congenital ocular malformations (anterior segment dysgenesis, congenital optic nerve malformation, and microphthalmia). Methods The NR2E1 coding region, 5′ and 3′ untranslated regions (UTRs), exon flanking regions including consensus splice sites, and six evolutionarily conserved non-coding candidate regulatory regions were analyzed by sequencing 58 probands with aniridia of whom 42 were negative for PAX6 mutations. Nineteen probands with anterior segment dysgenesis, one proband with optic nerve malformation, and two probands with microphthalmia were also sequenced. The control population comprised 376 healthy individuals. All sequences were analyzed against the GenBank sequence AL078596.8 for NR2E1. In addition, the coding region and flanking intronic sequences of FOXE3, FOXC1, PITX2, CYP1B1, PAX6, and B3GALTL were sequenced in one patient and his relatives. Results Sequencing analysis showed 17 NR2E1 variants including two novel rare non-coding variants (g.-1507G>A, g.14258C>T), and one novel rare coding variant (p.Arg274Gly). The latter was present in a male diagnosed with Peters’ anomaly who subsequently was found to have a known causative mutation for Peters’ plus syndrome in B3GALTL (c.660+1G>A). In addition, the NR2E1 novel rare variant Arg274Gly was present in the unaffected mother of the patient but absent in 746 control chromosomes. Conclusions We eliminated a major role for NR2E1 regulatory and coding mutations in aniridia and found a novel rare coding variant in NR2E1. In addition, we found no coding region variation in the control population for NR2E1, which further supports its previously reported high level of conservation and low genetic diversity. Future NR2E1 studies in ocular disease groups such as those involving retinal and optic nerve abnormalities should be undertaken to determine whether NR2E1 plays a role in these conditions.

The nuclear receptor 2E1 (NR2E1, also known as TLX) is involved in controlling proliferation in neural stem cells during brain and eye development. The role of NR2E1 in human disease is starting to be recognized as the genomic variation has been associated with bipolar disorder [24], and overexpression in gliomas correlates with decreased survival of patients with brain tumors [25]. NR2E1 is expressed early during eye morphogenesis in the eye field together with Pax6 [26], as well as in the optic cup and optic stalk [27,28]. Mice lacking Nr2e1 display brain and eye defects resulting from abnormal neural stem cell proliferation and depletion of the neural stem cell pool [29,30]. At the molecular level, there are different ways in which NR2E1 affects pathways involved in eye development. In mice, Nr2e1 represses Pax2 expression [28], which is required for optic cup and optic nerve development [31]. Pax2 and Pax6 mutually inhibit each other to define the retina and optic stalk boundaries [32] and dysregulation of this process negatively affects optic nerve development [33]. Thus, Nr2e1 may indirectly influence the expression of Pax6, a master regulator of eye development that results in various eye developmental disorders when mutated [34]. Evidence for such an interaction comes from Xenopus studies where Nr2e1 positively affects Pax6 levels [26]. Interestingly, the genetic interaction between Nr2e1 and Pax6 regulates the establishment of the dorsal-ventral cortical boundary in the mouse telencephalon [35]. In addition, Nr2e1 is involved in the retinoic acid pathway by potentiating the retinoic acid-mediated induction of the retinoic acid receptor beta 2 (RARβ2) promoter [36]. Interestingly, the retinoic acid pathway is involved in retinal [37] and anterior chamber morphogenesis [38,39]. Nr2e1 also has a non-cell autonomous role in activating the wingless signaling pathway to promote neural stem cell proliferation and self-renewal [40]. This pathway has many roles during eye development, including patterning of the ocular surface ectoderm [41]. Finally, NR2E3, the closest relative to NR2E1 in the human genome, causes enhanced S-cone syndrome and retinitis pigmentosa in humans [42][43][44]. Thus, due to the important role that NR2E1 plays during eye development, we hypothesize that NR2E1 may be involved in human eye disorders impacting a wide range of eye structures whose development depend on NR2E1 genetic interactors such as PAX2 and PAX6.
Overall, the NR2E1 locus is unusually highly conserved, reminiscent of the HOX cluster, and displays low genetic diversity among humans [24,45]. We have previously screened for NR2E1 mutations in patients with brain malformations [46,47] and bipolar disorder [24] but did not find any amino acid variations. However, 14 non-synonymous variants have now been reported in public databases: the Single Nucleotide Polymorphism database (dbSNP), the 1000 Genomes Project, and the NHLBI Exome Sequencing Project (ESP). Among these variants, six are predicted to confer amino acid substitutions that would affect protein function by sorting intolerant from tolerant ( SIFT) and polymorphism phenotyping (PolyPhen) scores; two were found in cancerous tissues in the heterozygous state, three were found in European-descendent ESP cohorts (comprising heart, lung, and blood diseases) in the heterozygous state, and one of unknown zygosity was found in European-descendent cohorts with atherosclerotic heart disease from the ClinSeq project [48].
Surprisingly, no cohort group comprised of individuals with a specific eye disorder has been screened for variation in NR2E1. To initiate such studies, we focused on sequencing NR2E1 in patients with aniridia but included patients with ASD, microphthalmia, and optic nerve malformations known not to harbor PAX6 mutations. We chose aniridia since we hypothesized that NR2E1 could alter PAX6 expression or functioning and ultimately lead to a phenotype resembling PAX6 haploinsufficiency. In this study, we identified several NR2E1 polymorphisms as well as a new amino acid variant in a patient diagnosed with Peters' anomaly (PA) who we subsequently found harbors a known causative mutation in B3GALTL. Sequencing of B3GALTL, CYP1B1, FOXC1, FOXE3, and PITX2 in the patient and his close relatives also revealed new variants in a subset of these genes. In conclusion, we did not find a causative mutation in NR2E1 that could explain aniridia. Fifty-eight probands were diagnosed with aniridia, one proband had Axenfeld-Rieger syndrome, one proband had The majority of patients were tested for PAX6 mutations and chosen to participate in this study after they were found negative. Thirty-one probands with aniridia were negative for PAX6 after sequencing and 11 probands with aniridia were negative for PAX6 after chromosomal analysis. N/A, not applicable. coloboma with congenital cataract, 12 probands had PA, five probands had Rieger syndrome, two probands had microphthalmia, and one proband had optic nerve malformation (Table 1). Slightly more than 70% of the samples collected had previously been examined for PAX6 pathogenic mutations and found to be negative using chromosomal analysis (11 aniridia samples [11 probands], one PA sample) and dideoxy fingerprinting or sequencing (33 aniridia samples [31 probands] and 21 samples of ASD, microphthalmia, and other disorders); see Table 1. The control group consisted of 282 individuals of Caucasian descent obtained from the Coriell Cell Repository, 188 of whom had been used in a previous study including 94 samples from individuals considered "neurologically normal" [24]. Ninety-four Caucasian patients diagnosed with Gilbert syndrome, a bilirubin disorder, also used in a previous study were included in this study as controls [24].

Patients and control individuals:
In addition, 22 unaffected relatives were included in the study to better assess the potential pathogenicity of the variants found. Eighteen were relatives of patients with aniridia, and four were relatives of patients with PA. NR2E1 sequencing analysis: Oragene DNA self-collection kits (DNA Genotek, Gaithersburg, MD) were used to collect saliva from patients and relatives during the 2007 Aniridia International Medical Conference (Memphis, TN). Genomic DNA was extracted using MoleStrips DNA Blood Kit (Lysaker, Norway) according to the manufacturer's instructions. Patient blood-purified DNA sent by collaborators was shipped and stored at 4 °C. Sequence analysis included bidirectional sequencing of the coding region, 5′ and 3′ untranslated regions (UTRs), exon flanking regions including consensus splice sites, and six evolutionarily conserved candidate regulatory non-coding regions using 20 polymerase chain reaction (PCR) amplicons as previously described   [47]. Human genomic NR2E1 (GenBank AL078596.8) sequence was used as the reference sequence. The numbering of NR2E1 variants was based on Antonarakis and the Nomenclature Working Group [49]. Every human NR2E1 variant found was confirmed by repeating the PCR and sequencing process. DNA samples from a subset of patients displaying NR2E1 variants g.14121C>G and g.14258C>T with unknown PAX6 genotype were subjected to targeted array comparative genomic hybridization analysis with exon-level resolution to identify deletions or duplications of one or more exons of PAX6 [50] with GeneDx (Gaithersburg, MD). In addition, these samples were analyzed for mutations with bidirectional sequencing of exonic regions of PAX6 (exons 1-13, the alternatively spliced exon 5a, and splice junctions) by GeneDx. B3GALTL, CYP1B1, FOXC1, FOXE3, and PITX2 were analyzed in patient 21,000 and his family for sequence variations by bidirectional sequencing of exons and at least 10 bp of flanking intron sequence. Sanger sequencing was performed using BigDye terminator kit version 3.1 and capillary electrophoresis on an ABI3130XL (Applied Biosystems, Carlsbad, CA). Subsequent data analysis was performed using SeqScape (Life Technologies, Carlsbad, CA). Primers for B3GALTL were previously described [11], and novel primers are depicted in Table 2. Forward primers had 5'-ACC CAC TGC TTA CTG GCT TAT C-3' and reverse primers 5'-GAG GGG CAA ACA ACA GAT GGC-3' added for sequencing of the PCR product (bolded, Table 2).

Database search and in silico analysis of variants: Single
Nucleotide Polymorphism Database was searched at dbSNP database (accessed July 2012). The 1000 Genomes database was searched at 1000 Genomes (accessed July 2012). The NHLBI Exome Sequencing Project (ESP) was searched at the Exome Variant Server, NHLBI Exome Sequencing Project (ESP), Seattle, WA (accessed July 2012). SIFT scores [51] were calculated using the online resource (accessed May 2011). PolyPhen scores [52] were calculated using the online resource (accessed July 2012).
Clinical assessment of patient 2,100: When patient 2,100 was an infant, a pediatric ophthalmological consultant performed bedside inspections, including assessment of visual acuity with large objects and preferential looking techniques. Examination of the exterior eye and eye movements was performed with a pencil light. Anterior segments were studied with a hand-held slit lamp, and visualization of the posterior segments by indirect ophthalmoscopy. Examinations under general anesthesia were performed with an operating microscope. Intraocular tension was assessed with applanation tonometry and a Schiötz tonometer. Retinal inspections were

GAGGGGCAAACAACAGATGGCTCCCTTTCTTTAGTGCCCACGACC
Bolded, sequences used for sequencing primers.  (Table 3). Only one variant was located in the coding region. Among the non-coding region variants, five were in the 5′-UTR, seven within intronic regions, and four within upstream conserved candidate regulatory elements. To explore whether the variants found represented polymorphisms, rare variants, or candidate mutations in NR2E1, we sequenced a control group of 376 unaffected individuals (752 normal chromosomes). Not all the amplicons were successfully sequenced for every control, and thus, the exact number of control chromosomes is depicted in Table 3.
Although most of our patients with aniridia were negative for PAX6 mutations, a fraction of the probands (11/58) had only chromosomal aberrations at the PAX6 locus analyzed, so point mutations and small deletion/insertions were not detected. Similarly, some patients sequenced for PAX6 exons may have intronic or upstream deletions that were not detected by the method used. In this way, our aniridia PAX6 negative group might have been overall smaller than 42 probands, thus reducing the power of our study.
The rare variant g.-1507G>A was located in a conserved element and was not previously reported but was also found in an unaffected relative of the patient with aniridia and thus was not a strong candidate for a causative mutation. However, two rare variants had not been previously reported and not found in the control population (Table 3); variant g.14258C>T was located in intron 7 in a patient with aniridia, and variant g.14121C>G (Arg274Gly) was located in exon 7 in a patient diagnosed with PA. We further sequenced PAX6 in these patients and found a known causal mutation [53,54] cooccurring with the variant g.14258C>T, which suggested that there was no functional significance for this NR2E1 variant. However, we did not find a PAX6 mutation in the male patient 21,000 harboring the variant g.14121C>G (Arg274Gly). In summary, we did not find any candidate mutations in NR2E1 in patients with aniridia but found one candidate mutation in a patient diagnosed with PA whom we characterized further as described below.
Clinical characteristics of patient 21,000: The patient, a boy, was the third child of a 40-year-old woman after six pregnancies, three of which were terminated by spontaneous abortion. The child was delivered by spontaneous birth in gestational week 38 with a low birthweight (1,775 g [<first percentile]) and birth length (40 cm [<first percentile]). The placenta was small with one-third infarction. No neonatal asphyxia was noted. Immediately after birth, the patient's large head circumference and corneal clouding were observed. Intracranial ultrasound showed intraventricular hemorrhage grade 3 with dilation of the ventricular system. A ventriculoperitoneal shunt was necessary to control his head circumference. Pediatric follow-up showed pronounced growth retardation. At three years of age, the bone age was retarded by two years. A laparoscopic examination established right testicular agenesis at six years of age. At 12 years, the beginning of puberty was noted, and his height had reached 126 cm (<third percentile). Puberty-suppressing treatment and growth hormone treatment were initiated despite normal hormone values to improve his final height. At 15 years, his height was 149 cm (<third percentile), and his weight 52.8 kg (20 th to 50 th percentile). He had normal proportions between the upper and lower trunk as well as extremities with normal hands and feet. He showed normal facial characteristics and had normal teeth and normal umbilicus. His fine and gross motor skills were appropriate for his age. His psychomotor development was described as normal by several examinations, the last one at the age of five. No renal failure was suspected, and no ultrasound exam of the kidneys was done. The patient had a normal karyotype, and no sign of inborn metabolic diseases in blood and urine.
When the patient was four days old, a corneal opacity on both eyes was noted during an ophthalmic examination, and a tentative diagnosis of Peters' anomaly was made. He was visually alert and had no nystagmus. At the age of six weeks, examination under general anesthesia disclosed a corneal lenticular contact with thread-like structures from the pupillary margin to the posterior lens surface. When the patient was 14 months old, binocular visual acuity of 20/200 was assessed by preferential looking. Reexamination under general anesthesia showed corneal diameters (right/left) of 11/11 mm, axial lengths of 19.8/20.8 mm, and intraocular tension of 11/11 mmHg. Gonioscopy showed dysgenesis of the iridocorneal angle with a fine membrane covering the peripheral part of the iris root, and drag on the peripheral iris. The lenses were clear, and indirect ophthalmoscopy showed normal optic nerve heads, normal retinal vessels and pigmentation, and no sign of persistent hyaloid vessels. Both eyes had normal diameters, large central corneal opacities with central thinning, and clear peripheries. Therapy-resistant glaucoma developed in both eyes and was complicated by keratopathy, nearly collapsed anterior chambers, and dense cataracts. At age 13, he was virtually blind and used Logtext and Braille in school.  Genetic assessment of patient 21,000: To comprehensively study patient 21,000, we sequenced additional candidate genes. This patient was originally diagnosed with PA, but careful review of clinical information revealed short stature and developmental delay resembling Peters' plus syndrome (PP). Thus, we screened the patient for additional genes known to be involved in the development of PA (CYP1B1, PITX2, FOXC1, and FOXE3) and PP (B3GALTL). During this work, we identified a known homozygous pathogenic variation c.660+1G>A in B3GALTL [11] indicative of PP (Table 4).
In addition, we found three novel non-pathological variants and nine known variants in B3GALTL, FOXC1, and FOXE3 (Table 4). Subsequently, we sequenced B3GALTL in patient 21,000's mother, father, and sister, and found the B3GALTL c.660+1G>A variation in the heterozygous state in all of them.
We then explored the possibility that the phenotype of patient 21,000 might be the result of a combination of mutations in B3GALTL and NR2E1 by further characterizing the NR2E1 rare variant g.14121C>G (Arg274Gly). The sequence trace of this variant showed a double C/G peak, indicative of heterozygosity and thus the presence of a Wt arginine (Arg) and a variant glycine (Gly) in NR2E1 at amino acid 274 (Figure 1). To better understand the biochemical and possible biologic consequences of the amino acid change, we considered the SIFT score, which was 0.01, suggestive of no tolerance for this amino acid substitution. In addition, homology-Basic Alignment Search Tool analysis depicts a high (>90%) NR2E1-protein conservation among vertebrates and 100% identity at Arg274. NR2E3 protein is also highly (>70%) conserved and has 100% identity at Arg309, which aligns with NR2E1 Arg274. Furthermore, a database search for NR2E1 coding variants revealed the Arg274Gln variant (dbSNP, rs148906882), found in a melanoma sample and thus of potential biologic significance [55]. We also screened for the Arg274Gly variant in the relatives of patient 21,000, including the mother, father, and sister (Figure 1), and found that the mother was positive for variant Arg274Gly but presented with no phenotypic eye abnormalities even after detailed reexamination. These results suggest that variant Arg274Gly did not contribute to the phenotype in patient 21,000.

DISCUSSION
NR2E1 is a candidate for human congenital ocular malformation based on its role in mouse eye development, and interaction with key eye developmental genes such as Pax2 and Pax6 as well as prominent signaling pathways that regulate eye morphogenesis such as wingless and retinoic acid. Based on these data, we undertook the first screening for NR2E1 mutations focused on human eye disorders. A patient population with congenital eye disorders enriched for lack of mutations in PAX6 was screened for sequence variation in functional regions of NR2E1 including candidate regulatory and coding regions. We extended the characterization of several known polymorphisms, and identified one novel rare variant in a conserved element (g. -1507G>A). In addition, we found one novel rare intronic variant (g.14258C>T) and one novel rare coding variant (g.14121C>G; p. Arg274Gly) not present in the control group. The latter represents one of the few amino acid changes found in NR2E1, all with unknown functional consequences, despite past substantial efforts to identify coding variants with sequencing-based mutation screening [24,46,47]; thus, we focused our further studies on this variant.
The novel rare NR2E1 coding variant was found heterozygous in a patient diagnosed with Peters' anomaly; the variant results in a substitution from Arg to Gly in amino acid 274 (Arg274Gly). Substantial evidence suggests that this amino acid change alters NR2E1 protein functioning: 1) the high conservation of Arg274 not only in NR2E1 but also in NR2E3 (Arg309) and the association of the NR2E3 variant, Arg309Gly, with eye disease [56]; 2) the low SIFT score indicating the substitution would not be tolerated; and 3) the possible clinical relevance of the Arg274Gln variant found in melanoma tissue [55], which, interestingly, has a SIFT score of only 0.04. However, patient 21,000 also harbored a known causative homozygous mutation in B3GALTL, and his phenotypically normal mother was heterozygous for the NR2E1 Arg274Gly variant. Although the patient does not seem to have typical PP due to the lack of facial dysmorphic features [57], it is unlikely that the NR2E1 variant has a role in improving this condition. Thus, we conclude that the Arg274Gly variant does not cause disease in the heterozygous state, which is in accordance with studies in mice where homozygous loss-of-function of Nr2e1 is required for brain phenotypes [58]. However, the potential remains that this variant could be found in a future patient contributing to the phenotype in a homozygous or compound heterozygous state.
In conclusion, we have eliminated a major role for NR2E1 regulatory and coding mutations in aniridia. In addition, the lack of coding region variation we have found in the control population for NR2E1 further supports the high level of conservation and low genetic diversity known for this gene [24,45]. These genomic characteristics also argue that most changes in the coding region have important biologic consequences. Thus, future studies in other ocular disease groups are well justified, and we propose that diseases involving retinal defects or optic nerve malformations should constitute the next research focus.